The Inhibition Effects Of Flavonoids On α-glucosidase And Nonenzymatic Glycation

At present,diabetes mellitus(DM)especially type 2 diabetes mellitus and its complications posed an increasingly serious threat to human health.α-Glucosidase hydrolyze oligosaccharides to release glucose,eventually leading to postprandial hyperglycemia.Therefore,suppression the activity of α-glucosidase to delay the release of glucose can reduce the postprandial hyperglycemia from the source,it is a approach for the treatment of DM;The increase of blood sugar levels lead to the acceleration of nonenzymatic glycation which would produce the advanced glycation end products(AGEs).Researches show that AGEs are associated with a series of diabetic complications.Since the α-glucosidase inhibitors and nonenzymatic glycation inhibitors currently clinical used would cause certain side effects in long-term use,so it is particularly important to search for "multi-target" effective drugs that is safe with little side effects and can inhibit both α-glucosidase and nonenzymatic glycation.Flavonoids are widely distributed in various plants and herbs with little side effects and various pharmacological effects.Screening effective α-glucosidase inhibitors and nonenzymatic glycation inhibitors from flavonoids or other natural plant active components become the focus of current study.In this study,α-glucosidase and nonenzymatic glycation were used as targets,and inhibition kinetics and interaction mechanisms of three flavonoids morin,apigenin and galangin to α-glucosidase were investigated by the multispectroscopic methods combined with molecular docking;Isobolograms were built to study the interaction between myricetin and agigenin,myrecetin and morin in the inhibition of α-glucosidase;Bovine serum albumin(BSA)-fructose was used as the model for in vitro nonenzymatic glycation to investigate the inhibitory effects of galangin,apigenin,morin and the combination of them on the three stages of nonenzymatic glycation,and then analyzed the interaction between two flavonoids.The main research contents and results are as follows:1.The inhibtion effects and ihibition kinetics of morin,apigenin and galangin on α-glucosidase were investigated.The results showed that morin,apigenin and galangin exhibited strong α-glucosidase inhibitory ability,the half inhibitory concentration(IC50)were(4.48 ± 0.04)× 10-6 mol L-1,(1.05 ± 0.05)× 10-5 mol L-1 and(2.75 ± 0.05)× 10-5 mol L-1,respectively,the order of inhibition effects was morin > apigenin > galangin,and all stronger than positive control acarbose((4.02 ± 0.05)× 10-4 mol L-1),it may be related to the hydroxylation at position 3 of C ring and the increasing number of hydroxyl groups in B ring.Morin and galangin were mixed type α-glucosidase inhibitors,and apigenin was noncompetitive inhibitors.The values of inhibition contants Ki of morin,apigenin and galangin were(1.68 ± 0.3)× 10-6 mol L-1,(1.07 ± 0.07)× 10-5 mol L-1 and(4.33 ± 0.4)× 10-6 mol L-1.2.The fluorescence spectroscopy,circular dichroism(CD)combined with molecular docking were applied to study the interaction mecanisms of morin,apigenin and galangin on α-glucosidase.The results shown that three flavonoids can interact with α-glucosidase to form ground complexes all driven by hydrophobic interaction and hydrogen bonding,resulting in the endogenous fluorescence quenching of enzyme,there was only one binding site on α-glucosidase for morin,apigenn and galangin.The binding distance r between morin,apigenin,galangin and α-glucosidase were 2.52 nm,2.77 nm and 4.08 nm,r(27)8 nm and 0.5R0(27)r(27)1.5R0,suggesting the high probability of energy transfer between α-glucosidase and three flavonoids.The results of synchronous fluorescence and CD indicated that the interaction of three flavonoids to α-glucosidase leading to the conformational changes of enzyme.Apigenin and galangin changed the microenvironment of tyrosine(Tyr)and tryptophan(Trp)residues in α-glucosidase,and the addition of morin,apigenin and galangin resulted to a content decrease of α-helix,from 30.1% to 25.4%,from 38.1% to 31.7%,from 37.1% to 29.8%,respectively.Molecular simulation results showed that morin and apigenin were binding near the active site,and the binding induced the conformational changes of α-glucosidase which blocked the channel to prevent the access of substrate to active site,weakening the catalytic activity of α-glucosidase.Galangin inserted into the active central of α-glucosidase,occupying the activity site of α-glucosidase to lower the catalytic activity of α-glucosidase.3.Isobolograms were built to study the inhibitory effects of myricetin and agigenin,myrecetin and morin on the activity of α-glucosidase,and analyzed the interaction between two flavonoids.The experimental results shown that the relative potency(R)between myricetin and agigenin was a constant,the additive isoboles were linear,at 50% inhibition effect level,when the concentration of myricetin fixed at 1.5 × 10–5 mol L–1 or apigenin kept at 9.0 × 10–6 mol L–1,the interaction between myricetin and agigenin was an additive interaction,at 70% inhibition effect level,when the concentration of myricetin was maintained at 2.25 ×10–5 mol L–1,indicating an antagonistic interaction,all the other study combinations were synergistic interaction.Myrecetin and morin shared a same maximal effect but different Hill coefficients and the relative potency was variable,so the additive isoboles were nonlinear,at 50% inhibition effect level,the concentration of morin kept at 3.5 ×10–6 mol L–1 and at 70% inhibition effect level,myricetin was maintained at 2.25 ×10–5 mol L–1,indicating the additive interaction between myrecetin and morin,all the other study combinations were synergistic interaction.These results showed that the combination of myricetin with apigenin or moirn exhibited a synergistic effect at low concentrations,whereas it tended to exhibit additive or antagonistic interaction with the increase of concentration.4.BSA-fructose was used as the model for in vitro nonenzymatic glycation to investigate the inhibitory effects of galangin,apigenin,morin and the combination of them on the three stages of nonenzymatic glycation,and then analyzed the interaction between two compounds.The study found that three flavonoids had a slight inhibitory effect on fructosamine(early stage products of nonenzymatic glycation)and dicarbonyl compounds(middle stage products of nonenzymatic glycation),and showed a strong inhibitory ability on fluorescent advanced glycation end products AGEs.The inhibitory effects of three flavonoids on the three stages were stonger than that of the positive control aminoguanidine AG;Among them,the inhibitory ability of apigenin was strongest,and the ability of galangin and morin was similar.The addition of galangin,apigenin and morin to the nonenzymatic glycation system resulted in the maximum emission wavelength of fluorescent AGEs had a red shift for 15 nm,8 nm and 22 nm,respectively.It may suggesting a increase of the polarity of the fluorophore microenvironments.The combination of galangin and apigenin,galangin and morin,apigenin and morin showed a stronger inhibitory effects on fructosamine,dicarbonyl compounds and fluorescent AGEs(the representative products for three stages of nonenzymatic glycation)compared to used alone.But the combinations of two flavonoids were just additive interaction rather than synergistic interaction.